Scheiper-Welling et al. BMC Medical Genetics
https://doi.org/10.1186/s12881-020-01170-3(2020) 21:227

RESEARCH ARTICLE

Open Access

Characterization of an N-terminal Nav1.5
channel variant – a potential risk factor forarrhythmias and sudden death?
Stefanie Scheiper-Welling1†, Paolo Zuccolini2†, Oliver Rauh2, Britt-Maria Beckmann3, Christof Geisen4, Anna Moroni5,
Gerhard Thiel2 and Silke Kauferstein1*

Abstract
Background: Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to anumber of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) anddilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified theheterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present
study, we aimed to investigate the functional impact of the variant to clarify the medical relevance.
Methods: Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed
functional characterization experiments using patch-clamp technique.
Results: Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel
functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5
current over the entire voltage window.
Conclusion: The results support the assumption that the detected sequence aberration alters Nav1.5 channel
function and may predispose to cardiac arrhythmias and sudden cardiac death.
Keywords: Arrhythmia syndromes, Sudden death, Cardiac arrest, SCN5A, Functional characterization

Background
Cardiac sodium channels are responsible for the inward
depolarizing current (INa) in the initial phase of the cardiac action potential. The cardiac expressed gene SCN5A
encodes the pore-forming ion conducting α-subunit of
the voltage-gated sodium channel Nav1.5, which ishighly conserved among species [1–4]. The Na+ current
resulting from Nav1.5 is fundamental in initiation andpropagation of action potentials and is therefore responsible for cardiac excitability and conduction velocity [2].
* Correspondence: kauferstein@em.uni-frankfurt.de†Stefanie Scheiper-Welling and Paolo Zuccolini contributed equally to thiswork.
1Institute of Legal Medicine, Goethe University of Frankfurt, Kennedyallee
104, 60596 Frankfurt am Main, Germany
Full list of author information is available at the end of the article

In cardiomyocytes, Nav1.5 is preferentially localized atintercalated discs and at the T-tubular system [1].
The sodium channel Nav1.5 consists of four homologous domains (DI, DII, DIII and DIV), each consisting ofsix membrane spanning segments (S1 - S6). The positively
charged S4 segments act as voltage sensor and are responsible for the voltage-dependent ion channel activation.
The pore of the sodium channel is formed by the extracellular loops (P-loops) between S5 and S6 segments. The Nas well as the C- terminal region of the α-subunit arelocated in the cytoplasm [3–7].
Genetic aberrations in SCN5A have been associatedwith different types of arrhythmias and diseases including
Brugada syndrome (BrS), long-QT syndrome (LQTS),
sick-sinus-syndrome (SSS), atrial fibrillation (AF), dilated© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Scheiper-Welling et al. BMC Medical Genetics(2020) 21:227

cardiomyopathy (DCM) and non-cardiac diseases as forexample epilepsy. However, abnormalities in the sodium
channel can also lead to overlapping phenotypes, so called
overlap syndromes [3, 6, 8–10]. Loss-of-function mutations have been associated with Brugada syndrome,
whereas gain-of-function variants are known to cause long
QT syndrome type 3. Next to those electrical diseases,
loss- as well as gain-of-function variants were described incases of the structural heart disease dilated cardiomyopathy. However, the precise mechanism leading to the
DCM phenotype remains unclear [3, 10, 11]. The pathogenesis and clinical spectrum of SCN5A variants is quite
complex. Whereas one patient is showing a severe phenotype, another patient carrying the same genetic variant exhibits no clinical conspicuities. Phenotypes may even vary
within one family. In the worst case, sudden cardiac death(SCD) is the first sign of a hereditary disease. The variabledisease severity, which is most likely influenced by environmental and genetic factors, remains incompletely
understood yet [3, 10, 12].
In the present study, we identified the variant of
unknown significance c.316A > G, p.(Ser106Gly) in the
N-terminal region of the cardiac sodium channel Nav1.5
in a patient with survived cardiac arrest. Variants in the
N-terminus of Nav1.5 have been identified in patientswith BrS and LQTS. However, the role of the Nterminal region on trafficking, localization and regulation is not fully understood until now [13, 14]. In order
to better assess the relevance of the detected variant, we
performed functional studies in HEK293 cells.

Methods
Ethics statement

The study was conducted in accordance with the “Declaration of Helsinki”. The patient signed informed consent with approval for publication.
Genetic analysis

DNA was extracted from blood using salting out procedure. Genetic screening was carried out by means of
Next-generation sequencing (NGS). Library preparation
was performed using Nextera Flex Technology combined with the TruSight Cardio Panel (Illumina) consisting of 174 genes with known cardiac associations(supplementary information). Subsequent sequencing
was carried out on a MiniSeq System (Illumina). The
resulting reads were aligned to the human reference
genome GRCh37/hg19. Variant calling and further
evaluation was performed using the Software GensearchNGS (Phenosystems). Variants were filteredaccording to a pre-established prioritization protocol as
described previously [15]. Evaluation was mainly basedon presumed functional impact on the protein and allele
frequency. Genetic aberrations expected to disrupt

Page 2 of 9protein function and with minor allele frequency below
0.2% in public available databases (The Genome Aggregation and Exome Sequencing Project databases) were
further evaluated. Detected sequence variants wereassessed using common databases (as e.g. public population databases, Human Gene Mutation Database) andapplying in silico prediction tools (PolyPhen-2 [16],MutationTaster [17], SIFT [18], CADD [19]). The identified variant was confirmed by polymerase chain reaction(PCR) and standard direct sequencing by means of BigDye Terminator v1.1 chemistry and 3130xl Genetic
Analyzer (Applied Biosystems). The resulting data were
evaluated using SeqScape Software.
Mutagenesis

For site-directed mutagenesis, the pcDNA3.1-EGFP vector comprising the wild cDNA sequence of SCN5A(hH1a isoform) was used (kindly provided by N. Neyroud, PhD, Inserm-Sorbonne Université Paris, France).
The required sequence variant was induced using the
QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). The resulting constructs were verified by direct sequencing.
Electrophysiological measurements

For the functional characterization of the variant
p.S106G, the mutant as well as wild type Nav1.5 channels were expressed in human embryonic kidney (HEK)cells, cell line 293 as described previously [20]. To mimic
the heterozygous state of the patient, wildtype and mutant channels were also co-transfected in a 1/1 ratio(WT/MT). HEK293 cells were cultivated at 37 °C, 5%
CO2 and passaged by transferring them to 35 mm plates.
Transfection was performed by means of TurboFect
transfection reagent (Thermo Fisher Scientific, Waltham,USA) and 3 μg of Nav1.5 constructs in the pcDNA3.1EGFP vector. On the next day, cells were detached using
accutase (PAA, GE Health Freiburg, Germany). Four
hundred microliters were transferred to a new platecomprising 2 ml medium. Electrophysiological measurements were performed on single cells approximately 36
h following transfection using conventional patch clamp
technique. Current measurements were carried out atroom temperature in whole-cell configuration. Measurements and data acquisition were performed with an
EPC-9 Patch Clamp amplifier (HEKA, Lambrecht,
Germany) and the Patch Master (HEKA) software. Data
were low pass filtered at 10 kHz and sampled at 20 kHz.
Currents were measured in a bath solution composed of
135 mM NaCl, 4 mM KCl, 1 mM CaCl2, 2 mM MgCl2,
20 mM Glucose and 10 mM HEPES (pH 7.4, adjustedwith NaOH). The pipette solution consisted of 140 mM
CsCl, 5 mM NaCl, 4 mM Mg-ATP, 2 mM MgCl2, 5 mM
EGTA, 10 mM HEPES (pH 7.4, adjusted with CsOH). In

Scheiper-Welling et al. BMC Medical Genetics(2020) 21:227

Page 3 of 9order to measure the peak current amplitudes and determine current/voltage (I/V) relationships, the cells wereclamped from a holding potential of − 140 mV (100 ms)
to test voltages ranging from − 100 mV to + 50 mV in 5
mV steps. To analyze the steady-state inactivation, cells
were clamped from a holding potential of − 100 mV(100 ms) to test voltages ranging from − 140 mV to − 20mV by 10 mV increments (500 ms), followed by a 50 ms
test pulse to + 20 mV. For data analysis, we utilized Igor
Pro 6.03 software (WaveMetrics, Lake Oswego, OR).
For noise analysis, Nav1.5 WT and S106G channels
were activated 200 times by a depolarizing voltage stepfrom a holding potential of − 140 mV to − 20 mV or −40 mV. Current traces were imported into MATLAB to
calculate the mean current <I > (t), the current differences between successive traces ΔIn(t), the mean current
differences <ΔI>(t), and the variance of the current differences σ2(t). <I > (t), ΔIn(t), <ΔI>(t), and σ2(t) were calculated with the following equations [21]:hIiðtÞ ¼

1 XM
M

I ðtÞ

n¼1 n

ð1Þ

I n ðt Þ − I nþ1 ðt Þ
2
1 XM
ΔI n ðt Þ
hΔI iðt Þ ¼
n¼1
M−1
2 XM
σ 2 ðt Þ ¼
ðΔI n ðt Þ − hΔI iðt ÞÞ2n¼1
M−1

ΔI n ðt Þ ¼

ð2Þ
ð3Þ
ð4Þ

where n is trace number and M the total number oftraces collected. σ2 values were plotted against Imean andfitted with the following parabolic equation:
σ 2 ðhI iÞ ¼ i∙hI i −hI i2
þ σ b2Nð5Þ

where i is the single channel amplitude, N the total
number of channels, and σb2 the background noise. Data
were fitted with the constrain that σb2 is ≥0.
Data analysis

Results are reported as mean ± standard deviation (SD)of n experiments. An unpaired student’s t-test was applied to determine the statistical significance of the results obtained.

Fig. 1 Detection of the SCN5A variant p.(Ser106Gly) in a patient with aborted cardiac arrest. a Schematic representation of the Nav1.5 channel
and localization of the detected N-terminal variant. b Alignment of a partial N-terminal sequence among mammals. The affected amino acid isindicated in red. c Assessment of the genetic variant by in silico prediction tools

Scheiper-Welling et al. BMC Medical Genetics(2020) 21:227

Results
Clinical background and genetic analysis

Next-generation sequencing using a defined cardio panel
revealed the heterozygous SCN5A variant c.316A > G,p.(Ser106Gly), rs1331765859, in a patient with survived
cardiac arrest. This variant is localized in the cytoplasmic N-terminal region of the Nav1.5 channel (Fig. 1a).
No further potentially informative rare variant (MAF ≤0.2%) was detected. The male patient experienced sudden cardiac arrest at the age of 35 and was finally resuscitated after 53 defibrillations. Cardiological examination
revealed no distinct clinical findings and echocardiographic findings most likely pointed towards an incipient
dilative cardiomyopathy as the left ventricular ejection
fraction was initially slightly reduced (LV-EF 50–55%),
but normalized quickly. Hence, this assumption could
not be confirmed during follow-up investigations including cardiac magnetic resonance imaging (MRI). ECG
tracings did not show specific findings and a provocative
test with intravenous administration of the sodium channel blocker ajmaline in order to unmask a concealed
Brugada syndrome was performed without displaying adiagnostic Brugada type I-ECG pattern (Fig. 2). So thecause for the cardiac arrest of the patient remained unclear and a arrhythmogenic cardiomyopathy could not
be excluded. The patient stated that his father received
the diagnosis of a cardiomyopathy, but unfortunately, he
was not willing to provide more detailed clinical data
and refused genetic analyses. The patient is also father
of a toddler, but due to agitation, cardiologicalPage 4 of 9

examination (ECG) was not possible until now. In orderto prevent anxiety no genetic analyses will be performed prior to cardiological assesment due to missing
specific cardiological consequences.
The amino acid serine (p.Ser106) in the intracellular
N-terminal region of the Nav1.5 channel is highly conserved in mammals (Fig. 1b). The sequence variant is
not listed in the common databases Exome Variant
Server, ExAC (Exome Aggregation Consortium) or GnomAD (The Genome Aggregation Database) indicatingthat this is a very rare variant. It is only listed in theTOPmed database (Trans-Omics for Precision
Medicine). The frequency stated in this database is0.001% (in 125,568 alleles). Quite recently, the variant
has been described in a sudden unexpected death of a
31 year old woman [22]. Aberrations in adjacent regions(p.Arg104Gln; p.Arg104Trp; Asn109Lys) have been associated with Brugada Syndrome [13, 14, 23].
In order to further assess the possible impact of the variant, several in silico predictions were applied. All tools
were pointing towards a possible causative impact on theprotein (Fig. 1c). Due to missing functional as well as cosegregation studies, this variant was classified as variant of
unknown significance (VUS) according to the American
College of Medical Genetics (ACMG) standards [24].
Electrophysiological measurements in HEK293 cells

In order to examine the functional relevance of theNaV1.5 variant p.Ser106Gly, wild type (WT) or mutant(MT) Nav1.5 channels were expressed with an n-Fig. 2 ECG findings of the patient. a ECG taken 1 year previous to the cardiac event is showing sinus rhythm with a heart rate of 66/min, flat P
waves, PR interval 140 ms, QRS 100 ms, T inversion in lead III and QTc of 389 ms. b ECG from approximately 3 months after cardiac event underbeta-blocker therapy is showing sinus bradycardia with a heart rate of 38/min, flat P waves, PR interval 190 ms, QRS 110 ms and QTc of 365 ms

Scheiper-Welling et al. BMC Medical Genetics(2020) 21:227

terminal GFP-tag in HEK293 cells. The representative
current traces in Fig. 3a show that whole-cell patch
clamp recordings of GFP positive cells exhibit in allcases large, NaV-type, inward currents. When expressedat equivalent levels, the Nav1.5-S106G generated current
was higher relative to that of the WT channel (Fig. 3b).
At a reference voltage of − 25 mV the current density ofthe mutant exceeded the control value by a factor of 1.6(P < 0.05). The density of the Na+ current exceeded thatof the control by a factor of 1.3 and fell in between control and mutant when both were transfected together(Fig. 3c). Because of a high variability of the measured
current densities this difference is not significant.
The S106G mutation also exhibited small effects on
the voltage dependency of the channel. The V0.5 value

Page 5 of 9for voltage-dependent inactivation is shifted by 3.3 mV
positive relative to the WT channel (− 45.3 mV (N = 8)
versus − 39.9 mV (N = 9) respectively). The respective
value for voltage dependence activation is in the mutant
shifted negative by 5.4 mV (− 91.5 vs. -88.2; P < 0.05 Fig.
3d). Combined with the shift in the inactivation curve
this predicts a slight increase and right shift of the window current, which is available during excitation (Fig.
3d). This combination of elevated current density andshift of the voltage dependency of the channel is expected to elevate the Na+ load of cardiomyocytes. We
also determined the same parameters for activation andinactivation in conditions in which WT and MT channel
were expressed together (Tab. 1). The results show thatthe curves for activation and inactivation tend towards

Fig. 3 Nav1.5 S106G results in gain of function. a Typical Na+ currents in HEK293 cells transiently expressing NaV1.5 WT (top) or NaV1.5-S106G
mutant (down). Currents were elicited by clamping cells from holding voltage (− 140 mV, 100 mS) to test voltages (between + 50 mV and − 100
mV, 100 ms) and back to post clamp voltage (− 140 mV, 100 ms). b Mean current density/voltage relationships (±SE) from cells transfected withWT (black, n = 9) or S106G mutant (red, n = 8). c Peak currents at − 25 mV from (b) for WT (black) mutant (red) and data from cells co-transfectedwith WT and S106G mutant (blue, n = 12). d Boltzmann fits of voltage-dependent inactivation (left) and activation (right) for Nav1.5 WT (black/grey) and NavS106G mutant (red/brown). The mutation causes a positive shift in the V1/2 value for inactivation (− 88.2 ± 0.8 mV versus 91.5 ± 1 mV)
and a negative shift of the V1/2 value for activation (− 45.3 ± 0.9 mV versus − 39.9 ± 0.8 mV). The P value for a difference between V1/2 values ofthe WT and mutant < 0.05. Inset: estimated window currents for WT and mutant from (b). The colors of the line correspond to those in (c)Scheiper-Welling et al. BMC Medical Genetics(2020) 21:227

Page 6 of 9

those of the mutant. They are significantly different (P <0.05) from the reference values of the WT channel.
The increase in current density of the mutant relative
to the WT over the entire voltage spectrum could originate from an increase in unitary conductance or an increase in the number of channels. To examine whether
the mutation affects the unitary conductance we estimated the single channel amplitude by non-stationary
noise analysis [25] in cells expressing the WT channel,
the S106G mutant or both. For the noise analysis we
considered the dynamics of channel inactivation fromthe peak current to the baseline (Fig. 4a). To eliminate
unwanted changes in the leak conductance we considered for each isochrone the variance in currents between
couples of subsequent voltage steps rather than the variance of the absolute values (see materials and methods).
For each cell we plotted the variance values as a functionof the mean current. The bell-shaped distribution could
be fitted with the parabolic Eq. 5 (an example is reported
in Fig. 4b). The first derivative of the function at thecurrent minimum is a good estimate of the single channel amplitude i [25].
For the WT and S106G construct we performed anoise analysis for clamp steps from holding voltage of −140 mV to − 20 mV and − 40 mV. The data in Fig. 4
show that the estimated single channel amplitudes (i)and the respective unitary conductance values are allvery similar. The results of these experiments imply thatthe mutation has no impact on the unitary conductanceof Nav1.5. This result is not surprising considering thatthe mutation is far from the pore of the channel.

Discussion
Aberrations in the SCN5A gene have been described tocause disease by altered Nav1.5 structure, function or expression level. Various types of arrhythmias and disorders have been associated in the last decades withalterations in this gene. In our study, we describe a rare
SCN5A variant, which might represent a potential risk
factor for fatal arrhythmias and sudden cardiac death. In
the present case, we detected the N-terminal missense
variant p.(Ser106Gly) in a patient with aborted cardiac
arrest. During follow-up investigations, the patient did
not exhibit distinct clinical conspicuities. Quite recently,
this variant has been described in a case of sudden unexpected death in a 31 year old woman [22]. It was known
that she suffered from syncope, but showed no ECG abnormalities or diagnostic echocardiographic findings
prior to death.
The results of the present experiments support theview that the mutation has an impact on channel
function and that this could be causally related to life
threatening arrhythmias. The electrophysiologicalcharacterization shows that the amino acid replacement

Fig. 4 S106G mutation does not affect unitary channel conductance.
a Exemplary recordings of a HEK293 cell expressing the Nav1.5 WTchannel for noise analysis. Channels were activated 200 times bystepping membrane from holding voltage (1–40 mV, 100 ms) to test
voltage of − 20 mV (100 ms). Red arrows delimit the part of the
traces used for the analysis. b Example of noise analysis from one
cell expressing Nav1.5 WT channel. The variance between pulses isplotted as a function of the mean current value. The data werefitted with parabolic function (Eq. 5) yielding the unitary channel
conductance from the initial slope of the fit. c Average i values(mean ± SE) for WT and S106G channels at two different voltages: −20 mV (WT n = 3, black; S106G n = 4, red) and − 40 mV (WT n = 4,
black; S106G n = 4, red)in the N-terminus of the channel has a gain of functional
impact on the channel. The most obvious functional
gain is an increase in steady state current. This is functionally also relevant in a heterozygotic patient because

Scheiper-Welling et al. BMC Medical Genetics(2020) 21:227

mixed expression of WT and MT still elevates the steadystate current relative to the WT channel. The mutation
has also small impacts on the voltage dependent parameters with the effect that the window between activationand inactivation is slightly increased and right shifted. In
analogy to the effect of other mutants of Nav1.5 [5] thecombination of these parameters implies that the
Ser106Gly mutation could increase in this manner the
influx of Na+ and elevate the Na+ lode of cardiomyocytes. The electrical data do not yet explain the diseasephenotypes of the patient carrying the mutant. But they
underscore that the mutant causes some aberrant
functions, which could be causally linked to heartarrhythmias.
Previous studies have already reported a vast numberof mutations in the Nav1.5 channel [5, 26, 27]. The majority of these mutations are localized in the transmembrane domains of the protein, where they affect mostly
gating features of the channel. In a second group themutations are found, like in the present case, in cytosolic
domains and associated with signaling or interactions
with scaffolding proteins [28]. In the context of recent
publications the phenotype of the present mutant may
however be better explained by a third category namely
mutations in which an interaction of cytosolic domains
is important for channel trafficking [13, 14]. A contribution of phosphorylation/dephosphorylation of the critical
Ser106 to this mechanism seems not relevant. A bioinformatics analysis suggests that several surrounding
residues namely Thr101, Thr108 and Ser115 are potential targets of kinases while Ser106 is not [29].
It is interesting to note that the critical S106G mutation is located between two cationic amino acids R104
and R121 in the Nav1.5 channel [13]. Mutations of these
two amino acids exhibit the exact opposite effect of the
S106G mutation: they decrease the current density andshift the activation curve positive. As an explanation forthe decrease in channel density in the R104W and
R121W mutants a quality control mechanism was proposed [13]. According to this mechanism the N-terminiof Nav1.5-subunits interact in the ER independent on
the critical amino acids. When a mutation in one of thebinding partners is mis-folded as a result of a mutation,
the protein complex is retained in the secretory pathway
and degraded. This quality control in the secretory pathway could reduce trafficking of channel proteins to theplasma membrane. It is tempting to speculate that the
S106G mutation has the opposite effect in that it accelerates the quality control mechanism and augments inthis manner trafficking of the channels to the plasma
membrane. Such an increase in the number of channels
would be in agreement with the experimental data,
which show an increase in Nav1.5 current over the entire
voltage window.

Page 7 of 9

The underlying mechanisms of SCN5A mutations
leading to gain-of-function effects are mainly due to abnormalities in channel kinetics. Various studies describe,that gain-of-function variants can cause many individual
phenotypes, ranging from LQTS, DCM as well as unclear clinical phenotypes and in the worst case sudden
death. Frequently, multiple phenotypes, so called overlap
syndromes, are described for one and the same SCN5A
mutation, which could also be explained by different
electrophysiological properties of the channel in different areas of the heart. Furthermore, it is generally assumed, that there are other unknown mechanisms thatare for instance related to age, co-morbidities orenvironmental influences that contribute to genotypephenotype interactions. Identification of other modulators and genetic factors may help to understand how
alterations in SCN5A can lead to so many diverse clinical phenotypes [3, 12, 26].
Based on the electrophysiological data, the severe cardiac event of the patient, in whom the variant was detected and last, the fact that the variant was identified ina further sudden death case of a young woman, who did
not show any ECG abnormalities prior to death, conspicuously [22], the n-terminal variant has to be considered as a risk factor for fatal arrhythmias and sudden
death.

Conclusions
In the present study, we describe an electrophysiologicalcharacterization of the very rare sequence variant
Ser106Gly in the SCN5A gene leading to a gain-offunction impact on the sodium channel Nav1.5. As
described in previous studies, our data underline the assumption that the cytosolic n-terminal domain is probably important for channel trafficking. The results
indicate, that the exchange of Ser for Gly is not recognized by any quality control mechanism and that the
mutant is transported to the membrane in a preferredmanner.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01170-3.
Additional file 1. List of genes included in the TruSight Cardio Panel(Illumina).

Abbreviations
AF: Atrial fibrillation; BrS: Brugada syndrome; DCM: Dilated Cardiomyopathy;
ECG: Electrocardiogram; EGFP: Enhanced green fluorescent protein;
HEK293: Human embryonic kidney cells (cell line 293); LQTS: Long QT
syndrome; LV-EF: Left ventricular ejection fraction; MRI: Magnetic resonanceimaging; MT: Mutant; NGS: Next-generation sequencing; SCD: Sudden
cardiac death; SSS: Sick sinus syndrome; WT: Wild type

Scheiper-Welling et al. BMC Medical Genetics(2020) 21:227

Acknowledgements
We thank Isabelle Feinauer for her technical work during cloning
experiments.

Page 8 of 9

7.

8.
Authors’ contributions
SS-W designed the study, performed and evaluated genetic analyses andmutagenesis, interpreted the results and wrote the manuscript draft. PZ
performed functional experiments, interpreted and visualized functional data
and was involved in writing the manuscript. OR analyzed and interpreted
data. B-MB examined the clinical data of the patient. CG was involved in
genetic analyses. AM and GT interpreted data and wrote manuscript draft. SK
supervised the study and was involved in writing the manuscript. All authors
have read and approved the final manuscript.
Funding
This work was in part supported by grant from DLR (50 WB1838) to GT. SK is
supported by the Friedrich Flick Foundation. Open Access funding enabled
and organized by Projekt DEAL.
Availability of data and materials
The raw data are available upon request from the corresponding author. The
genetic variant detected during this study is available in the NCBI dbSNP
database, rs1331765859. The human reference genome GRCh37/hg19 wasused as reference data set in this study.
Ethics approval and consent to participate
The current study was approved by the ethical commission of the University
Hospital, Goethe University Frankfurt (protocol number E84/06). Written
informed consent to participate was obtained from the patient involved inthis study.
Consent for publication
Consent for publication of the clinical data was obtained from all of theparticipants.

9.

10.

11.

12.

13.

14.

15.

16.

Competing interests
The authors declare that they have no competing interests.

17.

Author details
1
Institute of Legal Medicine, Goethe University of Frankfurt, Kennedyallee
104, 60596 Frankfurt am Main, Germany. 2Department of Biology, Membrane
Biophysics, Technische Universität Darmstadt, Schnittspahnstrasse 3, 64287
Darmstadt, Germany. 31 Institute of Legal Medicine, Goethe University ofFrankfurt, Kennedyallee104, 60596 Frankfurt am Main, Germany. 4German Red
Cross Blood Center, Institute of Transfusion Medicine and
Immunohaematology, University Hospital Frankfurt, Frankfurt, Germany.
5
Department of Biosciences and CNR IBF-Mi, University of Milano, Via Celoria
26, 20133 Milan, Italy.

18.

19.

20.

21.

Received: 30 June 2020 Accepted: 10 November 2020
22.
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